Objectives: Belgium is located between countries with low (Netherlands) or high (France) levels of antibiotic resistance. Border provinces from Belgium afford a unique opportunity to look at the clonal structure of isolates with non-susceptibility to two or more antibiotic classes (multiple resistance) across time and place. The clonal distribution of S. pneumoniae was analyzed in a sample of isolates collected from 19972004 in Limburg which borders the Netherlands and West-Vlaanderen which borders France.

Methods: A sample of 190 S. pneumoniae isolates collected in the provinces of Limburg and West-Vlaanderen was selected at random from a larger Belgian surveillance set. For each isolate, PCR was performed on seven housekeeping genes based on the universal Multi Locus Sequence Typing methods. Internal fragments were sequenced, compared to published alleles, and assigned allele numbers. Allele numbers were used to generate allelic profiles, which were compared to published allelic profiles to determine a sequence type (ST). The eBURST algorithm in conjunction with phylogenetic trees was used to assign STs to clonal groups.

Results: The 190 isolates contain 95 unique STs which are divided into 24 clonal groups. More than half of all isolates are contained within the 8 largest clonal groups. The vast majority of the clonal groups occur in both Limburg and West-Vlaanderen, however, the frequency of clones in each province is different. For example, clonal group 17 (representing the PMEN clone Spain 23F-1) is more likely to be present in West-Vlaanderen (94%) than in Limburg (6%) as is clonal group 1 (representing the PMEN clone Spain 9V-3). The distribution of multiple resistance (MR) varies across provinces and across clones with West-Vlaanderen harbouring 77.4% of the MR and Limburg only 22.6%. The variation in clonal group proportions explains much of the variation in MR levels between the provinces.

Conclusions: Although the same clonal complexes of S. pneumoniae are circulating in both Limburg and West-Vlaanderen, the frequency of clones varies significantly between the two provinces. A higher proportion of clonal groups harbouring MR such as Spain 23F-1 and Spain 9V-3 is seen in the Belgian province of West-Vlaanderen which borders France than in the Belgian province of Limburg which borders the Netherlands. There is evidence of a flow of clones and resistance from West-Vlaanderen to Limburg.